Prevention of pulmonary hypoplasia and pulmonary vascular remodeling by antenatal simvastatin treatment in nitrofen-induced congenital diaphragmatic hernia

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Prevention of pulmonary hypoplasia and pulmonary vascular remodeling by antenatal simvastatin treatment in nitrofen-induced congenital diaphragmatic hernia

Martine Makanga, Hidekazu Maruyama, Celine Dewachter, Agnès Mendes Da Costa, Emeline Hupkens, Geoffrey de Medina, Robert Naeije, and Laurence Dewachter

Laboratory of Physiology and Pharmacology, Faculty of Medicine, Université Libre de Bruxelles, Bruxelles, Belgium Submitted 13 November 2014; accepted in final form 15 January 2015

Makanga M, Maruyama H, Dewachter C, Mendes Da Costa A, Hupkens E, de Medina G, Naeije R, Dewachter L.Prevention of pulmonary hypoplasia and pulmonary vascular remodeling by antenatal simvastatin treatment in nitrofen-induced congenital diaphragmatic hernia. Am J Physiol Lung Cell Mol Physiol 308: L672–L682, 2015. First published January 23, 2015;

doi:10.1152/ajplung.00345.2014.—Congenital diaphragmatic hernia (CDH) has a high mortality rate mainly due to lung hypoplasia and persistent pulmonary hypertension of the newborn (PPHN). Simva- statin has been shown to prevent the development of pulmonary hypertension (PH) in experimental models of PH. We, therefore, hypothesized that antenatal simvastatin would attenuate PPHN in nitrofen-induced CDH in rats. The efficacy of antenatal simvastatin was compared with antenatal sildenafil, which has already been shown to improve pathological features of PPHN in nitrofen-induced CDH.

On embryonic day(E) 9.5, nitrofen or vehicle was administered to pregnant Sprague-Dawley rats. On E11, nitrofen-treated rats were randomly assigned to antenatal simvastatin (20 mg·kg^⫺1·day^⫺1 orally), antenatal sildenafil (100 mg·kg^⫺1·day^⫺1 orally), or placebo administration from E11 to E21. On E21, fetuses were delivered by cesarean section, killed, and checked for left-sided CDH. Lung tissue was then harvested for further pathobiological evaluation. In nitrofen- induced CDH, simvastatin failed to reduce the incidence of nitrofen- induced CDH in the offspring and to increase the body weight, but improved the lung-to-body weight ratio and lung parenchyma structure. Antenatal simvastatin restored the pulmonary vessel density and external diameter, and reduced the pulmonary arteriolar remodeling compared with nitrofen-induced CDH. This was associated with decreased lung expression of endothelin precursor, endothelin type A and B receptors, endothelial and inducible nitric oxide synthase, together with restored lung activation of apoptotic processes mainly in the epithelium. Antenatal simvastatin presented similar effects as antenatal therapy with sildenafil on nitrofen-induced CDH. Antenatal simvastatin improves pathological features of lung hypoplasia and PPHN in experimental nitrofen-induced CDH.

lung hypoplasia; persistent pulmonary hypertension of the newborn;

sildenafil

DESPITE RECENT ADVANCES IN antenatal and neonatal intensive care, congenital diaphragmatic hernia (CDH) remains a life- threatening cause of severe respiratory failure in the newborn (12), mainly because of combined ventilatory deficiency and persistent pulmonary hypertension of the newborn (PPHN) (50). CDH results in lung hypoplasia and pulmonary vascular abnormalities, including pulmonary artery remodeling (42),

whose severity mainly determines the outcome in CDH (1, 55).

However, the pathogenesis of pulmonary epithelial and vascular anomalies in CDH remains largely unknown and the drugs available for the clinicians insufficiently efficacious.

Statins have been shown to exert beneficial effects in various experimental models of pulmonary hypertension (15, 36, 37).

Simvastatin reversed established monocrotaline-induced pulmonary hypertension in rats by inducing apoptosis of neointimal smooth muscle cells (37) and chronic hypoxia-induced pulmonary hypertension by inhibiting the Rho signaling pathway (16). Simvastatin also improved pulmonary hypertension induced by a combination of vascular endothelial growth factor (VEGF) receptor inhibition and chronic hypoxia exposure by inducing endothelial apoptosis (53). Statins exert, indeed, di- rect anti-inflammatory (3), antiproliferative, and proapoptotic effects on vascular wall cells (11, 36, 37, 53) and improve endothelium-dependent relaxation by increasing endothelial nitric oxide (NO) synthase (34) and by reducing endothelin expression (18, 19).

We therefore hypothesized that antenatal maternal administration of simvastatin might show benefits in experimental nitrofen-induced CDH in rats. The efficacy of antenatal simvastatin therapy was compared with antenatal sildenafil administration, which has already shown beneficial effects on experimental (29) and human (38) CDH.

METHODS

All procedures and protocols were approved by the Institutional Committee on Animal Welfare of the Faculty of Medicine at the Université libre de Bruxelles (Brussels, Belgium).

Animal model.Pregnant Sprague-Dawley rats (Janvier, Saint-Bar- thevin, France) were gavage fed with 100 mg herbicide nitrofen (2,4-dichloro-4-nitrodiphenil ether; Fluka, Deisenhofen, Germany) dissolved in 1.5 ml olive oil as vehicle at embryonic day (E) 9.5 (predicted term: E22). Control rats received olive oil vehicle only.

Animals were randomized into four groups as follows: control rats, nitrofen-treated rats, nitrofen ⫹ simvastatin (20 mg·kg^⫺1·day^⫺1)- treated rats, and nitrofen ⫹ sildenafil (100 mg·kg^⫺1·day^⫺1)-treated rats. The simvastatin dose was chosen on the basis of previous studies evaluating the pharmacokinetics of simvastatin (8), its safety in pregnancy (52), and its efficacy in other pregnancy complication (24).

The sildenafil dose was chosen on the basis of previous studies showing efficacy of sildenafil in nitrofen-CDH (29). Simvastatin and sildenafil were administered orally in food pellets from E11.5 to E21.5. On E21.5, the animals were anesthetized, and fetuses were harvested by cesarean section. Fetuses were weighed, anesthetized, assessed for left-sided CDH, and killed by exsanguination. After being weighed, lungs (n⫽17–20 in each group, from two different litters) were snap-frozen in liquid nitrogen and stored at ⫺80°C for pathobiological evaluation. After overnight fixation in 4% formol, Address for reprint requests and other correspondence: L. Dewachter,

Laboratory of Physiology and Pharmacology, Faculty of Medicine, Université Libre de Bruxelles, 808, Lennik Rd. 1070-Brussels, Belgium (e-mail: ldewacht

@ulb.ac.be).

First published January 23, 2015; doi:10.1152/ajplung.00345.2014.

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lungs (n⫽5 in each group, from two different litters) were embedded in toto in paraffin for histopathological evaluation. Because not all fetuses develop CDH, only those who developed CDH in nitrofen-, nitrofen ⫹simvastatin-, and nitrofen ⫹ sildenafil-treated rats were further analyzed.

Lung architecture and pulmonary artery morphometry. Five-mi- crometer serial sections were taken along the longitudinal axis of the lung lobes and stained with hematoxylin-eosin for overall morphology assessment. Morphometric analysis of the overall lung architecture was performed estimating the mean wall transaction length (Lmw) and the radial alveolar count (RAC), as previously described by Roubliova et al. (45) and Cooney et al. (6), respectively. The total surface of each lung section was virtually divided in up to 20 random nonoverlapping fields for morphometric lung parenchyma analysis. Pulmonary artery morphometry was evaluated as previously described (44). Only small pulmonary arteries with an external diameter (ED)⬍100 ␮m and a complete muscular coat were measured. Medial thickness (MT) was related to arterial size by the formula %MT⫽(MT/ED)⫻100 and was measured by counting at least 50 pulmonary arteries per lung lobe from each fetus. Pulmonary vascular density (number of pulmonary arteries with ED ⬍100 ␮m/mm²) was evaluated using at least 50 microscopic fields (in⫻100 total magnification) of pulmonary sections randomly selected. All analyses were performed with a LEICA ICC 50 HC Plan (Switzerland) in⫻100 total magnification by two independent investigators in a blinded manner. The mean value was used for analysis.

Real-time polymerase chain reaction. Total RNA was extracted from snap-frozen lung tissue using TRIZOL reagent (Life Technolo- gies, Carlsbad, CA) followed by a chloroform/ethanol extraction and a final purification using the QIAGEN RNeasy Mini kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA concentration was determined by standard spectrophotometric tech- niques, and RNA integrity was assessed by visual inspection of GelRed (Biotium, Hayward, CA)-stained agarose gels. Reverse tran- scription was performed using random hexamer primers and Super- script II Reverse Transcriptase (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions.

For real-time polymerase chain reaction, sense and antisense primers were designed, using the Primer3 program, forRattus norvegicus preproendothelin-1 (PPET-1), endothelin-converting enzyme 1 (ECE1), endothelin receptor type A (ETA) and B (ETB), endothelial (eNOS) and inducible (iNOS) nitric oxide synthase, bone morphogenetic protein type 2 receptor (BMPR2), gremlin1, inhibitor of DNA- binding 1 (Id1), Bax, Bcl-2, and hypoxanthine phosphoribosyltransferase 1 (HPRT1; used as housekeeping gene) mRNA sequences (Table 1). To avoid inappropriate amplification of residual genomic DNA, intron-spanning primers were selected when exon sequences were known. For each sample, amplification reaction was performed in triplicate using SYBRGreen PCR Master Mix (Quanta Biosciences, Gaithersburg, MD), specific primers, and diluted template cDNA.

Result analysis was performed using an iCycler System (Bio-Rad Laboratories). Relative quantification was achieved with the compar- ative 2^⫺⌬⌬C^tmethod by normalization with the housekeeping gene (HPRT1). Results were expressed as relative fold increase over the mean value of relative mRNA expression of the control group arbi- trarily fixed to one.

Protein extraction and dosage.Proteins were extracted from snap- frozen pulmonary tissue samples by homogenization in an appropriate amount of ice-cold homogenizing lysis buffer [Complete Mini, pro- tease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) in phosphate-buffered saline and 0.1% Triton X-100]. The homogenates were centrifuged at 4°C, and the supernatants were collected. The protein content of total lung extract homogenates was measured by the method of Lowry et al. (28), using 5–25␮l homogenate and bovine serum albumin as standard (2– 80␮g/assay).

Enzyme-linked immunosorbent assay for lung content evaluation in endothelin-1. Endothelin-1 protein levels were determined with an Endothelin-1 Quantikine enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN) in lung protein extracts (200␮g), according to the manufacturer’s instructions. Endothelin-1 concentra- tions were obtained by referring to a standard curve realized in parallel. Results represented the mean values of two separate measurements preformed in duplicate.

Quantification of pulmonary total nitrate/nitrite levels. Because levels of more stable NO metabolites (nitrites and nitrates) have been used to measure indirectly the levels of NO in biological samples, total nitrate/nitrite (NOx) levels were determined using the Total Nitric Oxide Assay (R&D Systems) according to the manufacturer’s instructions. Briefly, lung protein extracts (200␮g) were ultrafiltered through a 10,000 molecular weight cutoff filter to eliminate proteins.

After reduction of nitrate to nitrite by nitrate reductase, total nitrite level was measured spectrophotometrically at 540 nm after incubation with Griess reagent at room temperature for 10 min. Pulmonary NOx

levels were obtained by referring to a standard curve realized in parallel and expressed as micromoles per gram total protein. Results are represented as the mean values of two separate measurements preformed in duplicate.

Terminal deoxynucleotidyl transferase dUTP nick-end labeling staining.Apoptotic cells were detected by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining using the ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit (catalog no.

Table 1. Sense and antisense primers used for RTQ-PCR analysis in rat tissue samples

Genes Primer Sequences

Preproendothelin-1

Sense 5=-CAGACCAAGGGAACAGATGC-3=

Antisense 5=-ACGCCTTTCTGCATGGTACT-3=

Endothelin-converting enzyme 1

Sense 5=-GCCCACCAAGAACGAGATT-3=

Antisense 5=-GACCCCGATACCAAAGT-3=

Endothelin receptor type A

Sense 5=-CTGGTGGCTCTTTGGATTCT-3=

Antisense 5=-GCTCCCATTCCTTCTGTTGA-3=

Endothelin receptor type B

Sense 5=-CGATTGTATCATGCCTCGTG-3=

Antisense 5=-GGGACCATTTCTCATGCACT-3=

Endothelial nitric oxide synthase

Sense 5=-GGTATTTGATGCTCGGGACT-3=

Antisense 5=-TGATGGCTGAACGAAGATTG-3=

Inducible nitric oxide synthase

Sense 5=-GTTTCCCCCAGTTCCTCACT-3=

Antisense 5=-CTCTCCATTGCCCCAGTTT-3=

Bone morphogenetic protein receptor type 2

Sense 5=-ATTGAGGGTGGGGTGGTAGT-3=

Antisense 5=-GTGAAACAAGGGTGCTGGTC-3=

Gremlin1

Sense 5=-CCTTTCTTTGCTCTCCCTGA-3=

Antisense 5=-TTGTCTCCCCACACTCTGAA-3=

Inhibitor of DNA binding 1

Sense 5=-CATGAACGGCTGCTACTCAC-3=

Antisense 5=-TGCAGTATCTCCACCTTGCTC-3=

Bax

Sense 5=-CGTGGTTGCCCTCTTCTACT-3=

Antisense 5=-TCACGGAGGAAGTCCAGTGT-3=

Bcl-2

Sense 5=-GTGGACAACATCGCTCTGTG-3=

Antisense 5=-CATGCTGGGGCCATATAGTT-3=

Hypoxanthine

phosphoribosyltransferase 1

Sense 5=-ACAGGCCAGACTTTGTTGGA-3=

Antisense 5=-TCCACTTTCGCTGATGACCAC-3=

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S7101; Chemicon, Temecula, CA) according to the manufacturer’s instructions. Negative control run without TdT enzyme and positive control pretreated with DNase I were tested. For each lung specimen, 10 different randomly chosen epithelial fields were examined. The epithelial apoptotic rate was calculated as the ratio of apoptotic nuclei (TUNEL-positive or brown nuclei) to total nuclei (brown⫹blue nuclei) (⫻100 to be expressed in percentage). The same procedure was used to evaluate the vascular apoptotic rate. All counts were performed by two independent investigators in a blinded manner. The mean value was used for analysis.

Statistical analysis. All data were expressed as means ⫾ SE.

Statistical analyses were performed using StatView 5.0 Software.

Comparisons of parameters between two groups were performed using one-way ANOVA. When theFratio of this analysis reached a critical P value ⬍0.05, comparisons were made with a modified two-tailed Student’s t-test.P values ⬍ 0.05 were considered to be statistically significant.

RESULTS

Effects of antenatal simvastatin on the incidence of CDH, body weight, and lung hypoplasia.In nitrofen-exposed litters, CDH was found in 19 of the 26 fetuses, thus 73% of the fetuses developed left-sided CDH. Neither antenatal simvastatin nor antenatal sildenafil treatment influenced the incidence of nitrofen-CDH (Fig. 1A).

Fetuses with nitrofen-induced CDH had a reduced body weight (by 22%) compared with controls. Antenatal simvastatin further reduced the body weight of nitrofen-CDH fetuses (by 37% compared with controls), whereas antenatal sildenafil did not (Fig. 1B). The lung-to-body weight ratio, a major indicator of lung hypoplasia, was lower (by 17%) in nitrofen- CDH compared with control fetuses (Fig. 1C). Antenatal simvastatin increased lung-to-body weight ratio in the nitrofen- CDH fetuses (by 12% compared with nitrofen-CDH fetuses), whereas antenatal sildenafil had no effect on the lung-to-body weight ratio (Fig. 1C). Lung weight did not change between

nitrofen-CDH fetuses treated or not with simvastatin (111⫾4 vs. 115⫾4 mg), suggesting that simvastatin-induced improvement in lung-to-body weight ratio could be (at least partly) the result of the decreased body weight.

Antenatal simvastatin improves lung architecture in fetuses with nitrofen-induced CDH.In fetuses with nitrofen-CDH, the thickness of the alveolar septa (assessed byLmw) was higher compared with controls (Fig. 2,AandC). Antenatal simvastatin reducedLmwin fetuses with nitrofen-CDH to similar levels as those observed in controls, whereas antenatal sildenafil failed to reduce significantly the Lmw(Fig. 2A).

RAC, an indicator of lung maturity, was lower in nitrofen- CDH fetuses. Antenatal simvastatin restored the RAC in nitrofen-CDH fetuses, as well as antenatal sildenafil. The RAC levels observed after antenatal simvastatin and sildenafil administration were similar to those of controls (Fig. 2B).

Antenatal simvastatin increases pulmonary vascular density and reduces pulmonary vascular remodeling in nitrofen-in- duced CDH.In fetuses with nitrofen-CDH, pulmonary arteries were less abundant and smaller (assessed by decreased ED;

Fig. 3, A and B). Antenatal simvastatin, as well as antenatal sildenafil, restored the pulmonary vascular density and the pulmonary artery ED in fetuses with nitrofen-CDH (Fig. 3,A andB).

As illustrated in Fig. 3, CandD, the medial wall thickness increased in the smallest pulmonary arteries (ED ⬍25 ␮m) from fetuses with nitrofen-CDH. Antenatal simvastatin, as well as antenatal sildenafil, decreased the medial wall thickness in these small pulmonary arteries from fetuses with nitrofen-CDH (Fig. 3, C and D). When the pulmonary artery MT was assessed in all pulmonary arteries (from all types of size), the resulting data did not show any differences between analyzed groups (data not shown). This suggests major remodeling of the smallest pulmonary arteries in fetuses with nitrofen-CDH.

0 1000 2000 3000 4000 5000 6000 7000

Control Nitrofen- CDH

Nitrofen- CDH + Simvastatin

Nitrofen- CDH + Sildenafil

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

Nitrofen- CDH + Sildenafil 0.0

0.5 1.0

Control Nitrofen-CDH Nitrofen-CDH + Simvastatin

Nitrofen-CDH + Sildenafil

A

B C

***

*** ***

***

CDH per Litter (in %)

Body Weight (in mg) Lung-to-Body Weight Ratio (in %)

***###

***$$$ *** ^# *

Fig. 1. Effects of antenatal simvastatin and sildenafil on the incidence of congenital diaphragmatic hernia (CDH), body weight, and the lung-to-body weight ratio. The proportion of CDH (A), fetal body weight (in mg;B), and the lung weight-to-body weight ratio (C) was evaluated in each litter from control (white bars), nitrofen-induced CDH (CDH; black bars), nitrofen-induced CDH treated with simvastatin (light gray bars), and sildenafil (dark gray bars) groups. eNOS, endothelial nitric oxide synthase; HPRT1, hypoxanthine phosphoribosyltransferase 1; iNOS, inducible nitric oxide synthase; NOx, nitrites plus nitrates. Values are expressed as means⫾SE.

*0.01 ⬍ P ⬍ 0.05 and ***P ⬍0.001 vs.

control fetuses; #0.01 ⬍ P ⬍ 0.05 and

###P ⬍ 0.001 vs. nitrofen-CDH fetuses;

$$$P⬍0.001 vs. nitrofen-CDH treated with simvastatin fetuses.

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Antenatal simvastatin restores the expression of molecules regulating the pulmonary vasomotion in nitrofen-induced CDH. Lung gene expression of the precursor of endothelin-1, PPET-1, and ETA increased in fetuses with nitrofen-CDH, whereas lung expression of the ECE1 and ETBdid not change (Fig. 4, A–D). This was confirmed at the protein level, with increased lung protein expression of endothelin-1 in the lungs from fetuses with nitrofen-CDH (Fig. 4E). Expression of eNOS and iNOS, as well as NOxlevels, decreased in the lungs from nitrofen-CDH fetuses (Fig. 5,A–C). This strongly suggests an imbalance of vasoconstrictor/vasodilator expression in nitrofen-CDH, in favor of an increased vasoconstriction.

In nitrofen-CDH, antenatal simvastatin restored lung gene expression of PPET-1 and ETAto control lung expression (or even lower for PPET-1) (Fig. 4, A and C). This was also observed for antenatal sildenafil in nitrofen-CDH (Fig. 4,Aand C). In nitrofen-CDH, lung protein expression of endothelin-1 decreased after antenatal simvastatin and sildenafil treatments but remained higher than lung endothelin-1 expression in controls (Fig. 4E). Antenatal simvastatin, as well as antenatal sildenafil, restored lung gene expressions of eNOS and iNOS and lung NOxlevels in nitrofen-CDH to similar levels as those observed in controls (Fig. 5,A–C). This suggests restoration of the altered vasoactive balance after antenatal simvastatin and sildenafil administration in nitrofen-CDH.

Antenatal simvastatin restores BMPR2 signaling in nitrofen- induced CDH. Lung gene expression of BMPR2 and Id1, a target gene of the BMPR2 signaling, decreased in fetuses with nitrofen-CDH, whereas expression of gremlin1, a BMPR2 antagonist, increased (Fig. 6, A–C). Antenatal simvastatin restored BMPR2 and Id1 expression in the lungs of fetuses with nitrofen-CDH (Fig. 6,AandC), whereas lung expression of gremlin1 remained high after simvastatin administration (Fig. 6B). Antenatal sildenafil induced similar effects as antenatal simvastatin on lung expression of BMPR2, Id1, and gremlin1 (Fig. 6,A–C).

Antenatal simvastatin acts on the epithelial and the pulmo- nary vascular apoptosis. Nitrofen-CDH was associated with decreased lung expression of proapoptotic Bax mitochondrial member, whereas lung expression of antiapopototic Bcl-2 did not change (Fig. 7, A and B). The resulting proapoptotic Bax/Bcl-2 ratio was decreased in the lungs of fetuses with nitrofen-CDH (Fig. 7C). Antenatal simvastatin, as well as antenatal sildenafil, restored lung expression of proapoptotic Bax and of the proapoptotic Bax/Bcl-2 ratio to similar levels as those of the control fetuses (Figs. 7Aand 6C).

To assess whether the upregulation in upstream apoptotic mitochondrial pathways was accompanied by completion of apoptotic processes and to localize the areas where the apoptotic processes were mostly activated, TUNEL staining was

0 2 4 6 8 10 12

Mean Wall Transection Length (Lmw, in μm) Radial Alveolar Count (RAC)

0 10 20 30 40

B A

**

##

***

### ###

Control Nitrofen-CDH

Nitrofen-CDH + Simvastatin

Nitrofen-CDH + Sildenafil

C

Fig. 2. Effects of antenatal simvastatin and sildenafil on lung morphometry. Mean wall transaction length (Lmw, in␮m;A) and radial alveolar count (RAC;B) in the lungs from control (white bars;n⫽5), nitrofen-induced CDH (CDH; black bars;n⫽5), nitrofen-induced CDH treated with simvastatin (light gray bars;

n⫽5), and sildenafil (dark gray bars;n⫽5) groups. Data are expressed as means⫾SE. **0.001⬍P⬍0.01 and ***P⬍0.001 vs. control fetuses; ##0.001⬍ P ⬍ 0.01 and ###P ⬍ 0.001 vs. nitrofen-CDH fetuses. C: representative hematoxylin- and eosin-stained pulmonary sections obtained from control nitrofen-induced CDH, nitrofen-induced CDH treated with simvastatin, and with sildenafil fetuses at 50-fold magnification. Scale bars: 500␮m.

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performed (Fig. 7,DandE). As illustrated in Fig. 7,DandE, nitrofen-CDH was associated with decreased apoptosis within the epithelium and the pulmonary vascular wall. Antenatal simvastatin restored the epithelial apoptotic rate in the lungs from fetuses with nitrofen-CDH to similar levels as those observed in controls, whereas it failed to restore it in the remodeled pulmonary vessels from nitrofen-CDH (Fig. 7, D andE). The epithelial and pulmonary vascular apoptotic rates remained low in the lungs of nitrofen-CDH fetuses after antenatal sildenafil administration (Fig. 7,DandE).

DISCUSSION

The present results show that antenatal maternal administration of simvastatin improves characteristic features of lung hypoplasia and PPHN in experimental nitrofen-CDH. This includes improvement of lung parenchyma architecture and decreased pulmonary arteriolar remodeling that appear to be related to the restoration of endothelin and NO systems, BMPR2 signaling, and activation of apoptotic processes (mainly in the epithelium).

CDH remains one of the greatest challenges in perinatal medicine (50). Malformations observed in CDH include failure of both pulmonary alveolar and vascular development (13, 23, 35), responsible for severe respiratory failure and PPHN in survivors. Here, we used nitrofen-induced CDH, which is a well-established experimental model of CDH recapitulating the major pulmonary abnormalities observed in human CDH, including lung hypoplasia and pulmonary vascular remodeling (33). In this experimental model, we showed that antenatal simvastatin significantly attenuated lung hypoplasia and improved lung parenchyma maturation in fetuses with nitrofen- CDH. The alveoli in the hypoplastic lungs presented an imma- ture form with reduced alveolarization and thickened intra- alveolar septa, responsible for a reduced capillary-air interface, which is essential for gas exchange. Simvastatin significantly improved these features, which are essential for the fetal outcome in CDH.

The endothelial dysfunction characterized by the imbalance in the production of vasoconstrictors (e.g., endothelin-1) and vasodilators (e.g., NO) together with the remodeling of the

0 5 10 15 20 25 30 35 40

0 5 10 15 20 25 30

Nitrofen- CDH + Sildenafil 0

2 4 6 8 10 12

Vascular density

A

Medial Wall Thickness (in %)

External Diameter (in μm)

B

C

D Control Nitrofen-CDH

Nitrofen-CDH + Simvastatin

Nitrofen-CDH + Sildenafil

*

#

*

## #

*

Fig. 3. Antenatal simvastatin and sildenafil improve lung vascular density and remodeling. Vascular density (expressed as the no. of pulmonary arteries with external diameter⬍100␮m/mm²;A) and morphometry on pulmonary arterioles expressed as pulmonary arterial external diameter (B) and medial wall thickness percentage (%MT) vs. external diameter (C) in the lungs from control (white bars;n⫽5), nitrofen-induced CDH (black bars;n⫽5), nitrofen-induced CDH treated with simvastatin (light gray bars;n⫽5), and sildenafil (dark gray bars;n⫽5) groups. Data are expressed as means⫾SE. *0.01⬍P⬍0.05 vs. control fetuses; #0.01⬍P⬍0.05 and ##0.001⬍P⬍0.01 vs. nitrofen-CDH fetuses.D: representative hematoxylin- and eosin-stained pulmonary arterioles obtained from control, nitrofen-induced CDH, nitrofen-induced CDH treated with simvastatin, and sildenafil fetuses at 400-fold magnification. Scale bars: 100␮m.

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pulmonary arteries contributes to high pulmonary vascular resistance and to the development of pulmonary hypertension in fetuses with CDH (32, 47). Here, we showed pulmonary arteriolar remodeling with muscularization of preacinar arteries in fetuses with nitrofen-CDH. Antenatal simvastatin reduced pulmonary vascular remodeling by decreasing medial wall thickness in the smallest pulmonary arteries. This is consistent with previous studies showing that simvastatin prevented and reversed pulmonary hypertension and pulmonary vascular remodeling in different experimental models of pulmonary hypertension (14, 36, 37, 53).

Interactions between pulmonary airways and blood vessels have been shown to play critical roles in normal lung development (51). Inhibition of VEGF in rat fetuses led to arrested alveolar development (21, 54). Moreover, the NO signaling played a critical role in vascular growth during septation and in modulation of perinatal pulmonary vascular tone (40). Conse- quently, promoting vascular growth before birth may improve lung maturation in CDH. Here, we showed decreased pulmo-

nary vascular density and reduced pulmonary artery ED in fetuses with nitrofen-CDH, suggesting pulmonary vascular instability and defective lung angiogenesis in these pups. This was associated with reduced pulmonary expression of eNOS and iNOS, as previously described in the lungs from human (2) and experimental (39, 56) CDH. All of these altered features were largely improved by antenatal simvastatin treatment, which is consistent with previous studies showing that simvastatin promoted angiogenesis in vitro and in vivo (4, 25) and improved endothelium-dependent relaxation through the upregulation of eNOS expression and NO production (25, 26).

Moreover, eNOS has been shown to play a pivotal role in the transition of the pulmonary circulation from fetal to neonatal life (48). Therefore, the restoration of eNOS and iNOS expression by antenatal simvastatin treatment in nitrofen-CDH may correlate with improved pulmonary endothelial function and therefore with the potential positive outcome of these pups.

Endothelin is an endothelium-derived potent vasoconstrictor (32) that has been shown to play important roles in modulating

0.0 0.5 1.0 1.5 2.0

0.0 0.5 1.0 1.5

0.0 0.5 1.0 1.5 2.0 2.5

0.0 0.5 1.0 1.5

Control Nitrofen-CDH Nitrofen-CDH

+ Simvastatin

A

B

E

C

Relative gene expression (PPET-1:HPRT1)Relative gene expression (ECE1:HPRT1) Relative gene expression (ETA:HPRT1)Relative gene expression (ETB:HPRT1)

*

*_# ^#

*_#

#

##

*

#

D

Pulmonaryendothelin-1protein content (pg/mg total protein) **

##

##*

0 10 20 30 40 50

Fig. 4. Antenatal simvastatin and sildenafil attenuate imbalance between pulmonary vasoconstrictor and vasodilatator-endothelin signaling. Relative gene expressions of precursor (preproendothelin-1, PPET-1;A) and enzyme implicated in endothelin-1 maturation (endothelin-converting enzyme-1, ECE1;B), endo- thelin receptors type A (ETA;C) and B (ETB; D), as well as protein levels of endothelin-1 (in pg/mg total protein;E) in the lungs from con- trol (white bars; n ⫽ 12), nitrofen-induced CDH (black bars; n ⫽ 11), nitrofen-induced CDH treated with simvastatin (light gray bars;

n⫽10), and sildenafil (dark gray bars;n⫽10) groups. Data are expressed as means ⫾SE.

*0.01⬍P⬍0.05 and **0.001⬍P⬍0.01 vs.

control fetuses; #0.01⬍P⬍0.05 and ##0.001⬍ P⬍0.01 vs. nitrofen-CDH fetuses.

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eNOS (57) and BMPR2 signaling (31) in pulmonary vascular cells, contributing therefore to the pulmonary artery remodeling. It acts via two different receptors, the type A and B receptors (ETA and ETB). Present on vascular smooth muscle cells, ETA receptor mediates vasoconstriction, whereas ETB

receptor, expressed on both endothelial and vascular smooth muscle cells, mainly mediates vasodilatation. Here, we showed increased gene expressions of endothelin precursor and ETA

receptor, as well as increased endothelin-1 protein content in the lungs from nitrofen-CDH while ETB expression did not

change. This is consistent with previous studies showing up- regulated endothelin signaling in the lungs from experimental (9) and human (7) CDH and beneficial effects of dual ETA-ETB

antagonism on the regulation of pulmonary vascular tone in experimental CDH (49). As described above, nitrofen-CDH was associated with decreased lung expression of eNOS and iNOS, both responsible for decreased NOx production and consequently for vasodilator effects within the pulmonary vasculature. Antenatal simvastatin restored lung gene expressions of endothelin precursor, ETAreceptor, eNOS and iNOS,

0.0 0.5 1.0 1.5

A

Relative gene expression (eNOS:HPRT1)

0.0 0.5 1.0 1.5

B

Relative gene expression (iNOS:HPRT1)

C

* *

0 10 20 30 40 50 60 70

Pulmonarytotal Nox level (μmol/mg total protein)

**

Fig. 5. Antenatal simvastatin and sildenafil attenuate imbalance between pulmonary vasoconstrictor and vasodilatator-nitric oxide signaling. Relative gene expressions of eNOS (A), iNOS (B), and levels of NOx(C) in the lungs from control (white bars; n ⫽ 12), nitrofen-induced CDH (black bars;n⫽11), nitrofen-induced CDH treated with simvastatin (light gray bars;n⫽10), and sildenafil (dark gray bars; n ⫽ 10) groups. Data are expressed as means⫾SE. *0.01⬍P⬍0.05 and **0.001⬍P⬍0.01 vs. control fetuses.

Relative gene expression (Gremlin1:HPRT1)Relative gene expression (BMPR2:HPRT1) Relative gene expression (Id1:HPRT1)

A

B

C

* ^#

* *

*

#

*

0.0 0.5 1.0 1.5

0.0 0.5 1.0 1.5 2.0 2.5

Fig. 6. Bone morphogenetic protein (BMP) signaling pathway. Relative gene expression of bone morphogenetic protein type 2 receptor (BMPR2;A), bone morphogenetic protein antagonist gremlin1 (B), and bone morphogenetic protein target gene, the inhibitor of DNA-binding 1 (Id1;C), in the lungs from control (white bars;n⫽12), nitrofen-induced CDH (black bars;n⫽11), nitrofen-induced CDH treated with simvastatin (light gray bars;n⫽10), and sildenafil (dark gray bars;

n⫽10) groups. Data are expressed as means⫾ SE. *0.01 ⬍P ⬍0.05 vs. control fetuses;

#0.01⬍P⬍0.05 vs. nitrofen-CDH fetuses.

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as well as lung endothelin-1 protein content and NO level (assessed by nitrate plus nitrite levels) in fetuses with nitrofen- CDH, which suggests restoration of pulmonary endothelial function in these pups.

The exact mechanisms by which simvastatin improved PPHN and lung hypoplasia in CDH remain to be clarified.

However, simvastatin has been shown to enhance the expression of BMPR2 in vascular endothelial cells (20), contributing therefore to the endothelial function and the regression of vascular disease. Fluvastatin treatment of microvascular endothelial cells induced Id1 expression (41), suggesting upregu- lated BMPR2 signaling induced by fluvastatin treatment. Sim- ilarly, sildenafil has been shown to potentiate BMP signaling in pulmonary artery smooth muscle cells preventing the development of pulmonary vascular remodeling in experimental pulmonary hypertension (59). We previously reported that BMP/

BMPR2 signaling was downregulated in the lungs from fetuses with experimental nitrofen-CDH (30). Here, we showed that antenatal simvastatin and sildenafil treatment both restored BMP/BMPR2 signaling in the lungs of fetuses with nitrofen- CDH. Because BMP/BMPR2 is largely involved in regulating the balance between cell proliferation and apoptosis, antenatal simvastatin and sildenafil probably act on the epithelial and vascular cell proliferation/apoptosis balance through the restoration of the BMPR2 signaling.

Disturbed balance between cell proliferation and apoptosis contributes to the development of lung hypoplasia (22) and pulmonary vascular remodeling (5) associated with CDH induced by nitrofen in rats. Here, we showed decreased activation of apoptotic processes in the epithelial and vascular layers in the lungs of nitrofen-CDH fetuses. Antenatal simvastatin partly reversed this decreased activation of apoptotic processes

0 10 20 30 40 50

Relative gene expression (Bax:HPRT1) Relative gene expression (Bcl-2:HPRT1)

A B

Bax-to-Bcl-2ratio (geneexpression)

C

*

**

# #

0.0 0.5 1.0 1.5

0.0 0.5 1.0 1.5 2.0

0.0 0.5 1.0 1.5 2.0 2.5

0 10 20 30 40

Epithelial apoptotic rate (in %)

** ^# *

Control Nitrofen-CDH

Nitrofen-CDH + Simvastatin

Nitrofen-CDH + Sildenafil

Vascular apoptotic rate (in %)

** ***

# ***

#

Control Nitrofen-CDH Nitrofen-CDH+ Simvastatin Nitrofen-CDH + Sildenafil

Control Nitrofen-CDH E

Nitrofen-CDH

+ Simvastatin Nitrofen-CDH + Sildenafil

D

Fig. 7. Effects of antenatal simvastatin and sildenafil on lung activation of apoptotic processes in nitrofen-induced CDH. Induction of mitochondrial apoptotic pathway: relative gene expression of pro (Bax;A)- and anti (Bcl-2; B)-apoptotic mitochondrial members of Bcl-2 protein family, as well as proapoptotic Bax/Bcl-2 ratio (C) in the lungs from control (white bars;n⫽12), nitrofen-induced CDH (black bars;n⫽11), nitrofen-induced CDH treated with simvastatin (light gray bars;n⫽10), and sildenafil (dark gray bars;n⫽10) groups. Execution of apoptosis in the airway epithelium and in the pulmonary arteries:

representative terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling (TUNEL) immunostainings in pulmonary epithelial (D) and artery (E) sections from control (white bars;n⫽5), nitrofen-induced CDH (black bars;n⫽5), nitrofen-induced CDH treated with simvastatin (light gray bars;n⫽5), and sildenafil (dark gray bars;n⫽5) groups. Scale bars: 50␮m. Apoptotic rates (%) were evaluated as the ratio between the no. of TUNEL-positive cells (brown nuclei shown by arrows) and the total number of cells (brown⫹blue nuclei) in the airway epithelial layer (D) and the pulmonary artery medial layer (E). Data are expressed as means⫾SE. *0.01⬍P⬍0.05, **0.001⬍P⬍0.01, and ***P⬍0.001 vs. control fetuses. #0.01⬍P⬍0.05 vs. nitrofen-CDH fetuses.

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in the epithelium, but not in the vessels, while antenatal sildenafil did not affect epithelial and vascular apoptosis. Our data are consistent with simvastatin effects reported in the epithelium in an experimental model of lung ischemia-reper- fusion injury (58). However, we did not see any change in the percentage of vascular TUNEL-positive cells, even though MT was reduced after antenatal simvastatin treatment. Similarly, antenatal sildenafil did not alter epithelial and vascular apoptotic rates, while Lmw and MT were decreased. A possible explanation for these apparent discrepancies could be differences in proliferation rates and/or elimination of vascular cells through apoptotic processes already accomplished on E21.

Based on animal and human data, maternal administration of simvastatin during pregnancy seems to be safe for the fetuses (43, 46). The simvastatin dose tested in the present study was chosen on the basis of these previous studies performed in pregnant rats. Here, we found reduced fetal body weight induced by antenatal simvastatin, suggesting intrauterine growth retardation. This is consistent with previous studies showing reduced body weight, but no reduced offspring sur- vival at birth and during the neonatal period after antenatal administration of simvastatin (10, 17). We did not evaluate the infant morbidity, which could be related to this fetal growth retardation. This should be clearly addressed in the further clinical trials. Our results on antenatal sildenafil administration in nitrofen-CDH were consistent with those obtained by Luong et al. in terms of improved characteristic features of lung hypoplasia and PPHN in nitrofen-CDH (29). Antenatal simvastatin was as effective as antenatal sildenafil in terms of lung hypoplasia and PPHN improvement in experimental nitrofen- CDH. However, even if pulmonary morphological and biological data obtained after antenatal simvastatin treatment in nitrofen-CDH were encouraging, one major limitation of the present study remains the lack of evaluation of physiological parameters, including pulmonary artery pressure and lung compliance. Experiments testing the efficacy of simvastatin in larger experimental models of CDH should be performed.

Because CDH can be diagnosed early (at 22 wk of gestation) by usual ultrasound examination in humans, antenatal therapies are therefore feasible. By analogy to the current use of gluco- corticoid treatment used to induce the maturation of fetal lungs and prevent postnatal complications in the newborns (27), we reasoned that antenatal simvastatin, an already used medication in pregnant women for other complications (such as pre- eclampsia), could improve lung epithelial and vascular development in CDH. However, further clinical studies testing the timing, dosing, and toxicity of maternal simvastatin administration in fetuses with CDH are clearly needed to evaluate efficacy and potential side effects (including intrauterine growth retardation) of antenatal simvastatin.

In conclusion, the present study shows that antenatal simvastatin treatment improves lung structure and decreases pulmonary arteriolar remodeling in nitrofen-CDH as efficaciously as antenatal sildenafil treatment.

ACKNOWLEDGMENTS

We thank Robert De Decker for help with gavage feeding of pregnant rats.

GRANTS

This work was supported by grants from the “Fonds de la Recherche Scientifique Médicale” (grant no. 3.4637.09) and the Belgian Foundation for

Cardiac Surgery. M. Makanga is a doctoral fellow from the Fonds of the Medical Missionaries of Mary (Dublin, Ireland). C. Dewachter was a doctoral fellow (“Aspirant FNRS”) from the “Fonds National de la Recherche Scien- tifique” (Belgium). L. Dewachter is a postdoctoral fellow (“Chargé de Recher- che”) from the “Fonds National de la Recherche Scientifique” (Belgium).

DISCLOSURES

The authors have no conflict of interest to declare.

AUTHOR CONTRIBUTIONS

Author contributions: M.M., C.D., and L.D. conception and design of research; M.M., H.M., C.D., A.M.d.C., E.H., G.d.M., and L.D. performed experiments; M.M., H.M., A.M.d.C., E.H., G.d.M., and L.D. analyzed data;

M.M., C.D., E.H., G.d.M., R.N., and L.D. interpreted results of experiments;

M.M., C.D., and L.D. drafted manuscript; M.M., H.M., C.D., A.M.d.C., R.N., and L.D. approved final version of manuscript; C.D., R.N., and L.D. edited and revised manuscript; L.D. prepared figures.

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